Angiogenesis is a prominent characteristic of glioblastoma tumors (GBM) and is considered a promising therapeutic target. Currently available anti-VEGF therapy does not change overall survival, however, and the issue of potential differences in angiogenesis among the molecular subtypes of GBM has not been addressed. Our preliminary data support the concept that glioma stem cells (GSCs) play a key role in GBM-associated angiogenesis and they do so, at least in part, by enhancing the migration of endothelial cells (ECs). ECs isolated from GBM exhibit greatly enhanced migration as compared to ECs isolated from normal brain and this heighted motility is promoted by GSC-conditioned media (GSC-CM). The effects of GSC-CM on the ECs appear to be associated, in part, with upregulation of the pro-angiogenic molecule ephrin-B2 on the ECs based on array analysis and blocking studies using recombinant ephrin-B2-Fc and EphB4-Fc (the ephrin-B2 receptor). This suggests that factors secreted by GSCs, including VEGF, act by upregulating ephrin-B2. In addition, the data indicate that a direct interaction between GSCs and ECs further promotes EC motility and that this interaction is mediated, in part, by an interaction between integrin v3 on ECs and L1 cell adhesion molecule (L1CAM) on GSCs. Notably, as ephrin-B2 promotes the activation and signaling of VEGFR2 and as VEGFR2 and integrin v3 reciprocally activate each other, these two mechanisms could intersect resulting in ephrin-B2 indirectly activating integrin v3. Thus, we propose a hypothetical model in which angiogenesis in GBM is promoted by increased EC motility that is driven by GSC-secreted factors that upregulate pro- angiogenic molecules on EC and that this mechanism enhances the effects of a direct interaction between L1CAM on the GSCs and integrin v3 on the ECs. This hypothesis will be tested using GSCs and ECs isolated from GBM of the mesenchymal and pro-neural subtypes using recombinant proteins, antibodies and downregulation approaches to: (1) Determine whether GSC-secreted factors (including VEGF) promote EC migration, proliferation and tubulomorphogenesis and expression of pro-angiogenic molecules (including ephrin-B2 and EphB4) on ECs. (2) Determine whether the interaction of integrin v3 on ECs with L1CAM on GSCs transmits a signal promoting EC migration, proliferation and/or tubulomorphogenesis as determined by co-culture of ECs and GSCs; stimulation with GSC-CM; and injection of ECs and GSCs into brain slices followed by 2-Photon Laser Scanning microscopy and individual cell motility tracking. (3) The mechanism will be tested in vivo by determining if the absence/blocking of integrin v3 or ephrin-B2 on ECs and L1CAM on GSCs alters angiogenesis, tumor volume and/or survival using PDGF-B-driven GFP-GBM cells or shL1CAM- GFP-GBM cells propagated in immunocompetent integrin 3-mutant or control mouse brain. RELEVANCE: These studies have the potential to provide a paradigm shift in the understanding of tumor-associated angiogenesis in general and will suggest novel regimens for improved anti-angiogenesis therapy in GBM.